Examination of substrate-induced conformational changes in the Na+/glucose cotransporter.
Conformations of the Na+/glucose cotransporter were examined using tryptophan fluorescence and substrates to induce cotransporter conformational changes. Addition of Na+ but not K+ or TMA+ resulted in a saturable quenching of tryptophan fluorescence with a K0.5 for Na+ of 28 mM. In the presence of saturating Na+ concentrations, d-glucose but not l-glucose, fructose, or phlorizin resulted in a partial return of tryptophan fluorescence to approximately 70% of the substrate-free levels. This return of tryptophan fluorescence was a saturable function of d-glucose concentration with a K0.5 of 43 microM. The three conformations were compared with respect to their sensitivity to tryptophan quench reagents. Acrylamide quenching was unaffected by substrates. In contrast, I- quenching decreased 40% in the presence of Na+, while Cs+ quenching increased 64%. Addition of saturating d-glucose concentrations resulted in the return of I- quenching to 90% of the substrate-free values and reduced Cs+ quenching to substrate-free levels. In contrast, phlorizin did not mimic the effect of d-glucose on tryptophan fluorescence. These results are interpreted in terms of a second substrate-induced cotransporter conformational change which based on similar substrate specificities appears directly related to cotransporter-mediated Na+ and d-glucose transport.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
D000818:Animals; D005947:Glucose / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000648:ultrastructure*; D009005:Monosaccharides / Q000378:metabolism; D010695:Phlorhizin / Q000378:metabolism; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D009004:Monosaccharide Transport Proteins; D009005:Monosaccharides; D014364:Tryptophan; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose